非标记定量法是磷酸化蛋白质定量极具研究价值和应用潜力的发展方向，该方法目前存在的主要问题是可靠性差，如何简化样品处理流程，大幅提高磷酸化肽段的富集效率和覆盖面，并改善非标记定量的灵敏度、准确度和重复性，是磷酸化蛋白质组非标记定量研究有待解决的核心问题。申请人拟构建一种新型杂合金属亲和色谱介孔材料，介孔结构具有大的比表面积和临时“固定化酶反应器”作用，能大大提高酶解效率；且表面键合的金属氧化物和金属离子杂合了不同亲和色谱富集机理，提高了磷酸化肽段的富集面、富集容量和灵敏度。该材料整合了酶解和磷酸化肽段富集过程，可一步完成样品前处理过程，有效解决了由于磷酸化肽段富集过程样品损失造成非标记定量可靠性差的问题。申请人计划将该材料结合非标记定量法，建立“集成式”样品处理方法，从而实现磷酸化蛋白质组的快速准确定量分析，为磷酸化蛋白质组研究提出全新的定量分析策略。

Label-free quantitative method is a future direction of quantitative phosphoproteomics which is worthy of study and owning potential application. The main problems of this method are poor reliability, how to simplify the sample processing, improve the enrichment efficiency and coverage of phosphorylated peptide, and enhance the sensitivity, accuracy and reproducibility of quantitative results, is still the key problem of label-free quantitative method used in phosphoproteomics. This project intends to design and synthesis a new type of hybrid metal affinity chromatography mesoporous material. Firstly, the large specific surface area of mesoporous structure was used to form a temporary "immobilized enzyme reactor", which can greatly improve the enzymatic efficiency. Secondly, the metal oxidation and metal ions binding on the surface can combine the advantages of different enrich mechanism of affinity chromatography which can greatly improve the enrich coverage, capacity and the sensitivity of the phosphorylated peptides. The material integrates the enzymatic hydrolysis and phosphorylated peptide enrich process, and finish the pre-treatment process in one step, which effectively avoid the loss of phosphorylated peptide during enrichment and solves the problem of poor reliability of label-free quantitative. This project plans to use the synthesized material in label-free quantitative method to establish an integrated sample processing to realize rapid and accurate quantitative analysis of phosphorylated proteome, and provide a novel quantitative analysis strategy for phosphorylated proteomics research.